Part:BBa_K4387007

Nitric Oxide Sensing Genetic Circuit With Three Ribosomal Binding Sites

Usage and Biology

In the frame of our project, we wanted to further improve the construct BBa_K4387005 by adding two more ribosomal binding sites to see if we could achieve a higher GFP response.

Thus this composite part consists of the inducible pNorVβ promoter, superfolder GFP preceded by three strong ribosomal binding sites (BBa_K4387020 [2], BBa_B0029, BBa_B0034), the NorR regulator, and a double forward terminator. We chose a high-copy backbone from Twist Bioscience for this part. Due to the competitive binding of the activated and inactivated NorR on the promoter, we decided on this construct with a positive feedback loop that adjusted the levels of NorR based on the amount of nitric oxide present [1]. The presence of nitric oxide would activate pNorVβ to induce GFP and NorR expression. Thereby, we ensure that high amounts of NorR will be produced only when NO is present.

This construct was tested in the bacterial strain E.coli Nissle 1917.

Characterization

We measured the GFP expression upon NO induction with a plate reader over 16 hours. Below is the dose-response curve of pNorV Beta, measured in a plate reader. For all measurements, we used the following conditions:

• Overnight growth and experiment were done in minimal M9 medium supplemented with Ampicillin at 37°C Start of experiment in 96 well plate at an OD600 of 0.05.
• Settings for GFP measurements: excitation at 485nm, emission at 520nm.
• Every condition was measured over three technical and three biological replicates.
• GFP emission was normalized to OD600.

According to figure__, we could prove that the construct with two ribosomal binding sites and the codon-optimized NorR BBa_K4387006 had the highest GFP response.

Measurements

Figure 1: Induction response of the pNorVβ promoter to different DETA/NO concentrations with respect to time.

Figure 2: Dose response of the pNorVβ promoter at different DETA/NO concentrations.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 746
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

• [1] Xiaoyu J. Chen, Baojun Wang, Ian P. Thompson, and Wei E. Huang et al. Rational Design and Characterization of Nitric Oxide Biosensors in E. coli Nissle 1917 and Mini SimCells ACS Synthetic Biology 2021 10 (10), 2566-2578 DOI: 10.1021/acssynbio.1c00223